dp 13: erwinia amylovora - ippc · united states are distinct from the strains on other hosts...
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Produced by the Secretariat of the International Plant Protection Convention (IPPC)
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ISPM 27ANNEX 13
DP 13: Erwinia amylovora
27
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This diagnostic protocol was adopted by the Standards Committee on behalf of the Commission on Phytosanitary Measures
in August 2016.
The annex is a prescriptive part of ISPM 27.
International Plant Protection Convention DP 13-1
ISPM 27 Diagnostic protocols for regulated pests
DP 13: Erwinia amylovora
Adopted 2016; published 2016
CONTENTS
1. Pest Information ............................................................................................................................... 3
2. Taxonomic Information .................................................................................................................... 3
3. Detection ........................................................................................................................................... 3
3.1 Detection in plants with symptoms ................................................................................... 4
3.1.1 Symptoms .......................................................................................................................... 4
3.1.2 Sampling and sample preparation ..................................................................................... 4
3.1.3 Isolation ............................................................................................................................. 5
3.1.3.1 Isolation from symptomatic samples ................................................................................. 5
3.1.3.2 Enrichment-isolation ......................................................................................................... 6
3.1.4 Serological detection ......................................................................................................... 6
3.1.4.1 Enrichment-DASI-ELISA ................................................................................................. 6
3.1.4.2 Direct tissue print-ELISA.................................................................................................. 7
3.1.4.3 Immunofluorescence ......................................................................................................... 7
3.1.4.4 Lateral flow immunoassay ................................................................................................ 8
3.1.5 Molecular detection ........................................................................................................... 8
3.1.5.1 Controls for molecular tests .............................................................................................. 8
3.1.5.2 DNA extraction ................................................................................................................. 9
3.1.5.3 DNA amplification by PCR .............................................................................................. 9
3.1.5.4 General considerations for PCR ...................................................................................... 11
3.1.5.5 Real-time PCR ................................................................................................................ 12
3.1.5.6 Interpretation of results from PCR .................................................................................. 13
3.1.5.7 Loop-mediated isothermal amplification ........................................................................ 13
3.2 Detection in asymptomatic plants ................................................................................... 14
3.2.1 Sampling and sample preparation ................................................................................... 14
3.2.2 Screening tests ................................................................................................................. 15
4. Identification ................................................................................................................................... 15
4.1 Nutritional and enzymatic identification ......................................................................... 16
4.1.1 Biochemical characterization .......................................................................................... 16
4.1.1.1 Nutritional and enzymatic profiling ................................................................................ 16
4.1.1.2 Automated identification ................................................................................................. 17
4.1.1.3 Fatty acid profiling .......................................................................................................... 17
4.2 Serological identification ................................................................................................ 17
4.2.1 Agglutination ................................................................................................................... 17
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DP 13 Diagnostic protocols for regulated pests
DP 13-2 International Plant Protection Convention
4.2.2 Immunofluorescence ....................................................................................................... 17
4.2.3 ELISA ............................................................................................................................. 17
4.2.4 Lateral flow immunoassay .............................................................................................. 18
4.3 Molecular identification .................................................................................................. 18
4.3.1 PCR ................................................................................................................................. 18
4.3.2 Macro-restriction and pulsed field gel electrophoresis ................................................... 18
4.4 Pathogenicity techniques ................................................................................................. 18
5. Records ........................................................................................................................................... 19
6. Contact Points for Further Information .......................................................................................... 19
7. Acknowledgements ........................................................................................................................ 19
8. References ...................................................................................................................................... 19
9. Figures ............................................................................................................................................ 23
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1. Pest Information
Erwinia amylovora is the causal agent of fire blight, a disease that affects most species of the
subfamily Maloideae of the family Rosaceae (Spiraeoideae). It was the first bacterium described as the
causal agent of a plant disease (Burrill, 1883). E. amylovora is considered to be native to North
America and was first detected outside North America in New Zealand in 1920. Fire blight was
reported in England in 1957 and since then the bacterium has been detected in most areas of Europe
where susceptible hosts are cultivated. E. amylovora is now present in more than 40 countries. It has
not been recorded in South America and most African and Asian countries (with the exception of
countries surrounding the Mediterranean Sea), and it has been eradicated in Australia after one report
there (van der Zwet, 2004). It represents a threat to the pome fruit industry of all these countries (Bonn
and van der Zwet, 2000). Details on geographic distribution can be found in the European and
Mediterranean Plant Protection Organization (EPPO) Plant Quarantine Data Retrieval System (EPPO,
n.d.).
The most important host plants from both economic and epidemiological viewpoints are in the genera
Chaenomeles, Cotoneaster, Crataegus, Cydonia, Eriobotrya, Malus, Mespilus, Pyracantha, Pyrus,
Sorbus and Stranvaesia (Bradbury, 1986). The E. amylovora strains isolated from Rubus sp. in the
United States are distinct from the strains on other hosts (Starr et al., 1951; Powney et al., 2011b).
Fire blight is probably the most serious bacterial disease affecting Pyrus communis (pear) and Malus
domestica (apple) cultivars in many countries. Epidemics are sporadic and are dependent on a number
of factors, including favourable environmental conditions, sufficient inoculum level present in the
orchard and host susceptibility. The disease is easily dispersed by birds, insects, rain or wind
(Thomson, 2000). The development of fire blight symptoms follows the seasonal growth development
of the host plant. The disease begins in spring with the production of the primary inoculum from
bacteria overwintering in cankers (Thomson, 2000) causing blossom infection, continues into summer
with shoot and fruit infection, and ends in winter with the development of cankers throughout the
dormant period of the host (van der Zwet and Beer, 1995; Thomson, 2000).
2. Taxonomic Information
Name: Erwinia amylovora (Burrill, 1883) Winslow et al., 1920
Synonyms: Micrococcus amylovorus Burrill, 1883, Bacillus amylovorus (Burrill,
1883) Trevisan, 1889, “Bacterium amylovorus” [sic] (Burrill, 1883)
Chester, 1897, Erwinia amylovora f.sp. rubi (Starr et al., 1951)
Taxonomic position: Proteobacteria, Y subdivision, Enterobacteriales, Enterobacteriaceae
Common name: Fire blight (EPPO, 2013)
3. Detection
Diagnosis of fire blight can be achieved using isolation and serological and molecular tests. The assays
indicated below are recommended after having been evaluated in one or more of the following ring
tests: in 2003 in a Diagnostic Protocols for Organisms Harmful to Plants (DIAGPRO) project
involving ten laboratories (López et al., 2006); in 2009 in a European Phytosanitary Research
Coordination (EUPHRESCO) project involving five laboratories (Dreo et al., 2009); and in 2010 by
fourteen laboratories worldwide (López et al., 2010). The tests indicated in Figures 1 and 2 are the
minimum requirements for the diagnosis, but further tests may be required by the national plant
protection organization (NPPO), especially for the first report in a country. For example, serological
tests may facilitate a presumptive diagnosis of symptomatic plant material based on the detection of a
specific protein; however, an additional test based on a different biological principle should be used
for detection. In all tests, positive and negative controls must be included.
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DP 13-4 International Plant Protection Convention
In this diagnostic protocol, methods (including reference to brand names) are described as published,
as these defined the original level of sensitivity, specificity and/or reproducibility achieved. The use of
names of reagents, chemicals or equipment in these diagnostic protocols implies no approval of them
to the exclusion of others that may also be suitable. Laboratory procedures presented in the protocols
may be adjusted to the standards of individual laboratories, provided that they are adequately
validated.
3.1 Detection in plants with symptoms
The recommended screening tests are indicated in the flow diagram in Figure 1.
3.1.1 Symptoms
Symptoms of fire blight on the most common hosts such as P. communis (pear), M. domestica,
(apple), Cydonia spp. (quince), Eriobotrya japonica (loquat), Cotoneaster spp. (cotoneaster),
Pyracantha spp. (pyracantha) and Crataegus spp. (hawthorn) are similar and easily recognized. The
name of the disease is descriptive of its major characteristic: the brownish, necrotic appearance of
twigs, flowers and leaves, as though they had been burned by fire. The typical symptoms are the
brown to black colour of leaves on affected branches, the production of exudate, and the characteristic
“shepherd’s crook” of terminal shoots. Depending on the affected plant part, the disease produces
blossom blight, shoot or twig blight, leaf blight, fruit blight, limb or trunk blight, or collar or rootstock
blight (van der Zwet and Keil, 1979; van der Zwet and Beer, 1995).
In apple and pear trees the first symptoms usually appear in early spring when the average temperature
rises above 15 °C, during humid weather. Infected blossoms become soaked with water, then wilt,
shrivel, and turn orange or brown to black. Peduncles may also appear water-soaked, and become dark
green and finally brown or black, sometimes oozing droplets of sticky bacterial exudate. Infected
leaves wilt and shrivel, and entire spurs turn brown in apples and dark brown to black in pears, but
remain attached to the tree for some time. Upon infection young fruitlets turn brown but also remain
attached to the tree. Immature fruit lesions appear oily or water-soaked, become brown to black, and
often ooze droplets of bacterial exudate. Characteristic reddish-brown streaks are often found in the
subcortical tissues when the bark is peeled from infected limbs or twigs (van der Zwet and Keil, 1979;
Thomson, 2000). Brown to black slightly depressed cankers form in the bark of twigs, branches or the
trunk of infected trees. These cankers later become defined by cracks near the margin of diseased and
healthy tissue (Thomson, 2000).
Confusion may occur between fire blight and blight- or blast-like symptoms – especially in blossoms
and buds – caused by other pathogenic bacteria and fungi, insect damage or physiological disorders.
Other bacteria that cause fire blight-like symptoms include Erwinia pyrifoliae, the causal agent of
bacterial shoot blight of Pyrus pyrifolia (Asian pear) (Kim et al., 1999); Erwinia piriflorinigrans,
isolated from necrotic pear blossoms in Spain (López et al., 2011); Erwinia uzenensis, recently
described in Japan (Matsuura et al., 2012); other Erwinia spp. reported in Japan that cause bacterial
shoot blight (Tanii et al., 1981; Kim et al., 2001a, 2001b; Palacio-Bielsa et al., 2012); and
Pseudomonas syringae pv. syringae, the causal agent of blossom blast. A definitive diagnosis of fire
blight should always be obtained through laboratory analysis.
3.1.2 Sampling and sample preparation
Plant material should be analysed as soon as possible after collection, but may be stored at 4–8 ºC for
up to one week until processing. Precautions to avoid cross-contamination should be taken when
collecting samples, during transport and processing, and especially while isolating the bacterium or
extracting DNA.
The samples should be processed with a general procedure valid for isolation, serological tests and
polymerase chain reaction (PCR) analysis. The use of freshly prepared antioxidant maceration buffer
(polyvinylpyrrolidone (PVP)-10, 20 g; mannitol, 10 g; ascorbic acid, 1.76 g; reduced glutathione, 3 g;
phosphate-buffered saline (PBS), 10 mM, 1 litre; pH 7.2; sterilized by filtration) is required for
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successful enrichment, as indicated by Gorris et al. (1996). The samples can be processed also in
sterile distilled water or in PBS, pH 7.2 (NaCl, 8 g; KCl, 0.2 g; Na2HPO4·12H2O, 2.9 g; KH2PO4, 0.2 g; distilled water, 1 litre) but for direct isolation, immunofluorescence or PCR.
Plant parts (flowers, shoots, twigs, leaves or fruit) showing the most typical symptoms, and with
bacterial exudate if possible, are carefully selected. Material for processing is selected from the
leading edge of disease lesions. The plant tissue is cut into pieces of approximately 0.1–1.0 g, crushed
lightly in antioxidant maceration buffer, PBS or sterile distilled water (as described in the previous
paragraph) at 1:50 (w/v), left to stand for at least 5 min, and placed on ice for a few minutes. Triplicate
samples (1 ml each) of each macerate are transferred to sterile microcentrifuge tubes, with one tube
stored at –20 ºC for subsequent analysis by PCR and another tube’s contents adjusted to 30% glycerol
and stored at –80 ºC for confirmation testing, if necessary. The third tube is kept on ice for performing
enrichment before enzyme-linked immunosorbent assay (ELISA) or PCR, and isolation on selective
media (Figure 1). If immunofluorescence is to be performed (i.e. immunofluorescence analysis is
optional), the slides are prepared and fixed on the same day that the samples are macerated. The PCR
analysis should be performed as soon as is convenient, using the macerated sample stored at –20 ºC.
3.1.3 Isolation
3.1.3.1 Isolation from symptomatic samples
In general, plating on three media is advised for maximum likelihood of recovery of E. amylovora,
especially when samples are not in good condition. Depending on the amount and composition of the
microbiota of the sample, each medium can be more or less efficient. Three media (CCT, King’s B
and levan) have been validated in two ring tests, with levan having the highest plating efficiency.
When symptoms are very advanced or the environmental conditions after infection are not favourable
for bacterial multiplication, the number of culturable E. amylovora cells can be very low. Isolation
under these conditions can result in plates with few cells of the pathogen and that can be overcrowded
with saprophytic and antagonistic bacteria. If this is suspected, the sample should be re-tested and/or
enriched before isolation. The induction of the reversible viable but non-culturable state (VBNC) has
been described for E. amylovora in vitro using copper treatments and in fruits (Ordax et al., 2009), and
it can be the cause of false negative isolation results.The recipes for the recommended media are
described below:
- CCT medium is prepared in two parts. Part 1 consists of: sucrose, 100 g; sorbitol, 10 g; Niaproof,4 1.2 ml; crystal violet, 2 ml (solvent 0.1% ethanol); nutrient agar, 23 g; distilled
water, 1 litre; pH 7.0–7.2; sterilized by autoclaving at 115 ºC for 10 min. The autoclaved
medium is cooled to approximately 45 ºC. Part 2 consists of: thallium nitrate, 2 ml (1% w/v
aqueous solution); cycloheximide, 0.05 g; sterilized by filtration. Part 2 is added to 1 litre sterile
Part 1 (Ishimaru and Klos, 1984).
- King’s B medium consists of: proteose peptone no. 3, 20 g; glycerol, 10 ml; K2HPO4, 1.5 g; MgSO4.7H2O, 1.5 g; agar, 15 g; distilled water, 1 litre; pH 7.0–7.2; sterilized by autoclaving at
120 ºC for 20 min (King et al., 1954).
- Levan medium consists of: yeast extract, 2 g; bactopeptone, 5 g; NaCl, 5 g; sucrose, 50 g; agar, 20 g; distilled water, 1 litre; pH 7.0–7.2; sterilized by autoclaving at 120 ºC for 20 min.
Cycloheximide is added at 0.05 g/litre to King’s B and levan media when fungi are expected in the
isolation. Dilutions of 1:10 and 1:100 of each macerate are prepared in PBS (NaCl, 8 g; KCl, 0.2 g;
Na2HPO4·12H2O, 2.9 g; KH2PO4, 0.2 g; distilled water, 1 litre).
Preferably 100 µl of the macerates and their dilutions is spread, by triple streaking, in 130 mm plates,
or 50 µl is spread in standard 90 mm Petri dishes. Plates are incubated at 25 ºC for up to four days.
The final reading is usually taken at 72 h. Colonies of E. amylovora on CCT medium are pale violet,
circular, high convex to domed, smooth and mucoid, and they grow more slowly than on King’s B or
levan media. Colonies on King’s B medium are creamy white, circular and non-fluorescent under
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DP 13 Diagnostic protocols for regulated pests
DP 13-6 International Plant Protection Convention
ultraviolet (UV) light at 366 nm. Colonies on levan medium are white, circular, domed, smooth and
mucoid. Levan-negative colonies of E. amylovora have been reported (Bereswill et al., 1997).
Pure cultures are obtained from individual suspect colonies of each sample by dilution and streaking
onto King’s B medium. Presumptive colonies of E. amylovora are identified preferably by double
antibody sandwich indirect (DASI)-ELISA, PCR or by other appropriate tests (e.g. biochemical,
immunofluorescence, fatty acid profile), or by inoculating susceptible organs of any available
E. amylovora host to test pathogenicity, as indicated in section 4.
When analysing symptomatic samples, good correlation is expected between isolation,
immunofluorescence, enrichment-DASI-ELISA (section 3.1.4.1) and PCR.
In the 2003 and 2010 ring tests, the accuracy of isolation was 0.88 and 0.81 for King’s B, 0.92 and
0.89 for levan, and 0.92 and 0.95 for CCT media, respectively (López et al., 2006; M.M. Lopez,
personal communication, 2012). In the 2009 ring test, accuracy of isolation was 0.96 for CCT (Dreo
et al., 2009).
3.1.3.2 Enrichment-isolation
Enrichment is used to multiply the initial population of culturable E. amylovora in a sample and to
perform enrichment-DASI-ELISA or enrichment-PCR. It should be carried out before isolation (even
for symptomatic samples) when a low number of culturable E. amylovora cells is expected to be
present (e.g. for copper-treated samples, samples with old symptoms, samples collected during
unfavourable weather conditions for fire blight such as in winter). The enrichment step greatly
increases the sensitivity of DASI-ELISA. The use of two validated liquid media for enrichment – one
non-selective (King’s B) and one semi-selective (CCT) – is advised because the composition and
population size of the microbiota are unknown.
The tissue sample is macerated as described in section 3.1.2 and 0.9 ml is immediately dispensed into
each of two sterile 10–15 ml tubes (to ensure sufficient aeration) containing 0.9 ml of each liquid
enrichment medium (King’s B without agar, and CCT made with nutrient broth instead of nutrient
agar). The tubes are incubated at 25 ºC for 48–72 h without shaking. A longer incubation is
recommended when processing plant samples collected in winter. Both enrichment broths and
dilutions (1:10 and 1:100) prepared in PBS are spread onto CCT plates, by triple streaking, to obtain
isolated colonies. Plates are incubated at 25 ºC for 72–96 h. Final reading of the CCT plates is at 72 h
and must be followed by purification of colonies and identification.
The use of semi-selective medium for plating and dilution is advised because the enrichment step will
permit growth of the pathogen but will also allow abundant multiplication of other bacteria. The
accuracy of the enrichment isolation on King’s B and CCT was 0.97 in the 2010 ring test.
3.1.4 Serological detection
3.1.4.1 Enrichment-DASI-ELISA
A kit for enrichment-DASI-ELISA has been validated in two ring tests and is available commercially
from Plant Print Diagnòstics SL1. It is based on the mixture of two specific monoclonal antibodies
described in Gorris et al. (1996) and requires prior enrichment of the samples, as previously described.
The following protocol must be followed strictly for maximum accuracy. Before ELISA, the required
amount of the enriched extracts and controls is treated by incubation in a water bath at 100 ºC for
10 min. This treatment is necessary for optimum specificity. The boiled samples are processed (at
1 In this diagnostic protocol, methods (including reference to brand names) are described as published, as these
defined the original level of sensitivity, specificity and/or reproducibility achieved. The use of names of
reagents, chemicals or equipment in these diagnostic protocols implies no approval of them to the exclusion of
others that may also be suitable. Laboratory procedures presented in the protocols may be adjusted to the
standards of individual laboratories, provided that they are adequately validated.
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Diagnostic protocols for regulated pests DP 13
International Plant Protection Convention DP 13-7
room temperature) by ELISA on the same day (or stored at –20 ºC for subsequent analysis) following
the instructions provided by the manufacturer of the commercial kit.
The ELISA is negative if the average optical density (OD) reading from duplicate sample wells is 2×
the OD in the negative sample extract control wells (providing all negative control wells are lower
than 2× the average OD reading of the positive control wells).
Negative ELISA readings in positive control wells indicate that the test has not been performed
correctly and/or the reagents were not well prepared. Positive ELISA readings in negative control
wells indicate cross-contamination or non-specific antibody binding. In both cases, the test should be
repeated or a second test based on a different biological principle, such as PCR, should be performed.
In the 2003 and 2010 ring tests the accuracy of the DASI-ELISA was 0.79 and 0.82, respectively, for
enrichment in King’s B medium (King’s B-DASI-ELISA), and 0.83 and 0.77, respectively, for
enrichment in CCT medium (CCT-DASI-ELISA) (López et al., 2006, 2010).
3.1.4.2 Direct tissue print-ELISA
To make tissue prints, freshly cut plant sections are pressed carefully against a nitrocellulose
membrane. Prints are prepared for positive and negative controls. Printed membranes can be kept for
several months in a dry place at room temperature. A validated source of antibodies to E. amylovora
such as the Plant Print Diagnòstics SL kit1 should be used. To develop prints, the manufacturer’s
instructions should be followed. The prints are observed under low power magnification (×10 or ×20).
The test is positive when purple–violet precipitates appear in the sections of plant tissue that are
printed on the membrane and not in the plant tissue print of the negative control. If exudates or
colonies are printed they should appear violet when positive. The test is negative when no purple–
violet precipitates appear, as in the negative control.
3.1.4.3 Immunofluorescence
Immunofluorescence is a recommended alternative serological method, and it is easy to follow the
standard protocol (Anonymous, 1998). A validated source of antibodies to E. amylovora should be
used. Two commercial antibodies have been validated in one ring test: one monoclonal antibody is
available through Plant Print Diagnòstics SL1 and one polyclonal antibody is available from Loewe
Biochemicals1.
Immunofluorescence should be performed on fresh sample extracts fixed onto slides. Undiluted
macerates and dilutions of 1:10 and 1:100 in PBS are used to spot windows of the
immunofluorescence slides. The monoclonal or polyclonal antibody is used at the appropriate dilution
in PBS. The appropriate fluorescein isothiocyanate (FITC) conjugate is diluted in PBS: goat anti-
mouse for monoclonal antibody (GAM-FITC), and goat anti-rabbit (GAR-FITC) or anti-goat for
polyclonal antibody.
The test on a sample is negative if green fluorescing cells with morphology typical of E. amylovora
are observed in the positive controls, but not in the sample windows. The test on a sample is positive if
green fluorescing cells with typical morphology are observed in the positive controls and in the sample
windows, but not in the negative controls. As a population of 103 cells/ml is considered the limit for
reliable detection by immunofluorescence, for samples with >103 cells/ml, the immunofluorescence
test is considered positive. For samples with
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3.1.4.4 Lateral flow immunoassay
Two lateral flow devices are available commercially for rapid analysis of plant material: Ea AgriStrip
(Bioreba1) and Pocket Diagnostics (Forsite Diagnostics1). Following the manufacturers’ instructions
their accuracy in the 2009 and 2010 ring tests was 0.66 and 0.55, respectively, for Ea AgriStrip1 and
0.64 and 0.56, respectively, for Pocket Diagnostics1. These results were obtained for the detection of
E. amylovora in samples from 1 to106 c.f.u./g, but the accuracy was approximately 1.0 when analysing
samples with 105 to 106 c.f.u./g, the minimum number expected in symptomatic samples (López et al.,
2010). The kits are recommended for use only with symptomatic samples.
3.1.5 Molecular detection
Several PCR methods and one loop-mediated isothermal amplification (LAMP) protocol2, available
for the detection of E. amylovora, were evaluated extensively in ring testing by several laboratories
(Lopez et al., 2010; M.M. Lopez, personal communication, 2012). The specificity of some of these
methods has been evaluated by Powney et al. (2011a). Conventional PCR methods may be more
expensive and time consuming and usually require more training than serological methods, and for
these reasons, as well as the risk of contamination, they are not always appropriate for large-scale
testing. However, real-time PCR and some conventional PCR and nested PCR in one tube protocols
have provided highly accurate results and they are therefore recommended molecular methods. All
PCR assays should be performed using DNA extracted from the samples because of the high amount
of inhibitors of E. amylovora hosts, or from enriched samples, which have increased reliability of
detection.
3.1.5.1 Controls for molecular tests
For the test result obtained to be considered reliable, appropriate controls – which will depend on the
type of test used and the level of certainty required – should be considered for each series of nucleic
acid isolation and amplification of the target nucleic acid. For PCR a positive nucleic acid control, an
internal control and a negative amplification control (no template control) are the minimum controls
that should be used.
Positive nucleic acid control
This control is used to monitor the efficiency of the test method (apart from the extraction), and
specifically the amplification. Pre-prepared (stored) nucleic acid, whole genome amplified DNA or a
synthetic control (e.g. cloned PCR product) may be used.
Internal control
For conventional and real-time PCR, plant internal controls (e.g. a housekeeping gene (HKG) such as
COX (Weller et al., 2000) or 16S ribosomal (r)DNA (Weisberg et al., 1991)) should be incorporated
into the protocol to eliminate the possibility of PCR false negatives due to nucleic acid extraction
failure or degradation or the presence of PCR inhibitors.
Negative amplification control (no template control)
This control is necessary for conventional and real-time PCR to rule out false positives due to
contamination during preparation of the reaction mixture. PCR-grade water that was used to prepare
the reaction mixture is added at the amplification stage.
2 When using LAMP on a regular basis in an area that has a patent system such as Japan (patent no.s 3 313 358,
3 974 441 and 4 139 424), the United States (US6 410 278, US6 974 670 and US7 494 790), the European
Union (no.s 1 020 534, 1 873 260, 2 045 337 and 2 287 338), China (ZL008818262), the Republic of Korea
(patent no. 10-0612551), Australia (no. 779160) and the Russian Federation (no. 2 252 964), it is necessary for
users to obtain a licence from Eiken Chemical Co., Ltd before use to protect the intellectual property right.
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Positive extraction control
This control is used to ensure that target nucleic acid extracted is of sufficient quantity and quality and
that the target is detected. Nucleic acid is extracted from infected host tissue or healthy plant tissue
that has been spiked with the target.
The positive control should be approximately one-tenth of the amount of leaf tissue used per plant for
the DNA extraction.
For PCR, care needs to be taken to avoid cross-contamination due to aerosols from the positive control
or from positive samples. If required, the positive control used in the laboratory should be sequenced
so that this sequence can be readily compared with sequence obtained from PCR amplicons of the
correct size. Alternatively, synthetic positive controls can be made with a known sequence that, again,
can be compared with PCR amplicons of the correct size.
Negative extraction control
This control is used to monitor contamination during nucleic acid extraction and/or cross-reaction with
the host tissue. The control compromises nucleic acid that is extracted from uninfected host tissue and
subsequently amplified. Multiple controls are recommended to be included when large numbers of
positive samples are expected.
3.1.5.2 DNA extraction
Three DNA extraction methods – Llop et al. (1999), Taylor et al. (2001) and the REDExtract-N-Amp
Plant PCR Kit (Sigma-Aldrich1) – were evaluated in the 2009 ring test (Dreo et al., 2009) with four
PCR protocols with accuracies ranging from 0.67 to 0.76. The methods showed comparable results in
the 2010 ring test (Lopez et al., 2010), as indicated below in the accuracies given for the different PCR
methods. Their efficiencies did not improve after diluting the extracts 1:10, suggesting that few or no
inhibitors were present. Based on these findings, the Llop et al. (1999) extraction method is
recommended as it has been extensively tested in a number of countries and is cheap and easy to set
up in the laboratory.
DNA extraction according to Llop et al. (1999)
One millilitre of a sample macerate prepared according to section 3.1.2 and/or 1 ml enriched macerate
is centrifuged at 10 000 g for 5 min at room temperature. The supernatant is discarded, and the pellet
is resuspended in 500 µl extraction buffer (Tris-HCl pH 7.5, 24.2 g; NaCl, 14.6 g;
ethylenediaminetetraacetic acid (EDTA), 9.3 g; sodium dodecyl sulphate (SDS), 5 g; PVP-10, 20 g;
distilled water, 1 litre; sterilized by filtration) and incubated for 1 h at room temperature before
centrifugation at 4 000 g for 5 min. Approximately 450 µl supernatant is mixed with an equal volume
of isopropanol, inverted, and left at room temperature for 30 min to 1 h. The precipitated nucleic acid
is centrifuged at 10 000 g for 5 min, the supernatant is discarded and the pellet is air-dried. If there is
still a coloured precipitate (brown or green) at the bottom of the tube, this is carefully removed while
discarding the supernatant, thus obtaining a cleaner DNA pellet. The pellet is resuspended in 200 µl
water. It should be used for PCR immediately or stored at –20 ºC.
3.1.5.3 DNA amplification by PCR
There are many PCR primers and protocols described for E. amylovora detection and some have
shown specificity problems (Roselló et al., 2006; Powney et al., 2011a). The primers and protocols
validated in ring tests were those of Bereswill et al. (1992) and Llop et al. (2000), with or without
previous enrichment, in 2003; and those of Taylor et al. (2001), Stöger et al. (2006) and Obradovic
et al. (2007) in 2009 and 2010. The discovery of fully virulent E. amylovora strains without the
pEA29 plasmid (Llop et al., 2006) and experiences from different countries (Powney et al., 2011a)
indicate that two PCR protocols should be used: one with primers based on pEA29 sequences, and
another with primers targeting unique chromosomal sequences. If the PCR is negative with the
protocol based on the pEA29 primers and positive with the protocol based on the chromosomal
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primers, the PCR test can be considered as positive for E. amylovora. PCR can be carried out using the
primers and conditions validated in the ring tests, although amplification conditions should be
optimized for different thermocyclers.
PCR according to Bereswill et al. (1992)
The primers are:
A (forward): 5′-CGG TTT TTA ACG CTG GG-3′
B (reverse): 5′-GGG CAA ATA CTC GGA TT-3′
The targeted sequences are in the plasmid pEA29. The PCR mixture is composed of: ultrapure water,
17.4 µl; buffer 10×, 2.5 µl; MgCl2 50 mM, 1.5 µl; dNTPs 10 mM, 0.5 µl; primer A 10 pmol/µl,
0.25 µl; primer B 10 pmol/µl, 0.25 µl; and Taq DNA polymerase 5 U/µl, 0.1 µl. The extracted DNA
sample volume is 2.5 μl, and should be added to 22.5 μl of the PCR mix. The cycling parameters are a
denaturation step of 93 ºC for 5 min followed by 40 cycles of 93 ºC for 30 s, 52 ºC for 30 s and 72 ºC
for 1 min 15 s, with a final elongation step at 72 ºC for 10 min. The amplicon size is 900 base pairs
(bp) according to Bereswill et al. (1992), although variations in size can occur between 900 and
1 100 bp depending on the number of 8 bp repeats within the amplified fragment (Jones and Geider,
2001).
The accuracy was 0.51 in the 2003 ring test but increased to 0.74 and 0.78 after enrichment of the
samples in King’s B and CCT media, respectively (López et al., 2006).
PCR according to Taylor et al. (2001)
The primers are:
G1-F: 5′-CCT GCA TAA ATC ACC GCT GAC AGC TCA ATG-3′
G2-R: 5′-GCT ACC ACT GAT CGC TCG AAT CAA ATC GGC-3′
The targeted sequences are chromosomal. The PCR mixture is composed of: ultrapure water, 14.3 µl;
buffer 10×, 2.5 µl; MgCl2 50 mM, 0.75 µl; dNTPs 10 mM, 0.25 µl; G1-F 10pmol/µl, 1 µl; G2-R
10pmol/µl, 1 µl; and Taq DNA polymerase 5 U/µl, 0.2 µl. An extracted DNA sample of 5 μl is added
to 45 μl PCR mix. The cycling parameters are 95 ºC for 3 min followed by 40 cycles of 94 ºC for 30 s,
60 ºC for 30 s and 72 ºC for 1 min, with a final extension step at 72 ºC for 5 min, and cooling at 15 ºC.
The expected amplicon size is 187 bp.
The accuracy was 0.77 in the 2010 ring test using the Llop et al. (1999) DNA extraction procedure.
PCR according to Stöger et al. (2006)
The primers (from Llop et al., 2000) are:
PEANT1-F: 5′-TAT CCC TAA AAA CCT CAG TGC-3′
PEANT2-R: 5′-GCA ACC TTG TGC CCT TTA-3′
The targeted sequences are in the plasmid pEA29. Stöger et al. (2006) recommended this method be
used with DNA extracted using the REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich1). The PCR
mixture is composed of: ultrapure water, 5 µl; REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich1),
10 µl; PEANT1-F 10 pmol/µl, 0.5 µl; PEANT2-R 10 pmol/µl, 0.5 µl; and extracted DNA, 4 µl. The
cycling parameters are 95 °C for 5 min followed by 35 cycles of 95 °C for 15 s, 58 °C for 30 s and
72 °C for 45 s, with a final extension step at 72 °C for 5 min, and cooling at 15 °C. The expected
amplicon size is 391 bp.
The accuracy was 0.76 in the 2009 ring test and 0.72 in the 2010 ring test with the recommended DNA
extraction kit.
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PCR according to Gottsberger (2010) (adapted from Obradovic et al. (2007))
The primers are:
FER1-F: 5′-AGC AGC AAT TAA TGG CAA GTA TAG TCA-3′
rgER2-R: 5′-AAA AGA GAC ATC TGG ATT CAG ACA AT-3′
The targeted sequences are chromosomal. The PCR mixture is composed of: ultrapure water, 14.3 µl;
buffer 10×, 2.5 µl; MgCl2 50 mM, 0.75 µl; dNTPs 10 mM, 0.25 µl; FER1-F 10 pmol/µl, 1 µl; rgER2-R
10 pmol/µl, 1 µl; Taq DNA polymerase 5 U/µl, 0.2 µl; and extracted DNA, 5 µl. The cycling
parameters are 94 ºC for 3 min followed by 41 cycles of 94 ºC for 10 s, 60 ºC for 10 s and 72 ºC for
30 s, with a final extension step at 72 ºC for 5 min, and cooling at 15 ºC. The expected amplicon size
is 458 bp.
The accuracy was 0.76 in the 2009 ring test and 0.68 in the 2010 ring test using the DNA extraction
method described by Llop et al. (1999).
Nested PCR according to Llop et al. (2000)
The nested PCR of Llop et al. (2000) uses two sets of primers, which are combined in a single reaction
tube. Because of the different annealing temperatures of the primers the two PCRs are run
consecutively. The external primers are those designed by McManus and Jones (1995) and are based
on sequences of the pEA29 plasmid. The internal primers are those described by Llop et al. (2000).
The external primers are:
AJ75-F: 5′-CGT ATT CAC GGC TTC GCA GAT-3′
AJ76-R: 5′-ACC CGC CAG GAT AGT CGC ATA-3′
The internal primers are:
PEANT1-F: 5′-TAT CCC TAA AAA CCT CAG TGC-3′
PEANT2-R: 5′-GCA ACC TTG TGC CCT TTA-3′
The PCR mixture is composed of: ultrapure water, 36.25 µl; buffer 10×, 5 µl; MgCl2 50 mM, 3 µl;
dNTPs 10 mM, 0.5 µl; AJ75-F 0.1 pmol/µl, 0.32 µl; AJ76-R 0.1 pmol/µl, 0.32 µl; PEANT1-F
10 pmol/µl, 1 µl; PEANT2-R 10 pmol/µl, 1 µl; and Taq DNA polymerase 5 U/µl, 0.6 µl. A DNA
sample volume of 2 µl should be added to 48 µl PCR mix. The cycling parameters are a denaturation
step of 94 ºC for 4 min followed by 25 cycles of 94 ºC for 60 s and 72 ºC for 90 s. This first round
PCR is followed in the same thermocycler by a second denaturation step of 94 ºC for 4 min and 40
cycles of 94 ºC for 60 s, 56 ºC for 60 s and 72 ºC for 60 s, with a final elongation step at 72 ºC for
10 min. The expected amplicon size is 391 bp, although variations in size can occur.
The accuracy was 0.69 and 0.72 in the 2003 and 2010 ring tests, respectively, but increased after
enrichment to 0.84 (King’s B medium) and 0.86 (CCT medium) in the 2003 ring test, and to 0.79
(King’s B) and 0.88 (CCT) in 2010.
3.1.5.4 General considerations for PCR
The PCR protocols may need to be modified (optimized) when using different reagents or
thermocyclers.
After PCR amplification the presence of E. amylovora can be confirmed by sequencing the PCR
products or by restriction fragment length polymorphism (RFLP) analysis. The restriction pattern
observed in the amplicons obtained with the primers of Bereswill et al. (1992) or with the nested PCR
of Llop et al. (2000) can be used to confirm the specificity of the PCR analysis when compared with
the restriction pattern of a known control strain. Restriction digestion should be performed with the
endonucleases DraI and SmaI.
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The test on a sample is negative if the E. amylovora-specific amplicon of the expected size (and the
restriction enzyme pattern or amplicon sequence, when applicable) is not detected in the sample but is
detected in all positive controls. The test on a sample is positive if the E. amylovora-specific amplicon
of the expected size is detected, providing there is no amplification from any of the negative controls
and the restriction enzyme pattern or amplicon sequence (when applicable) is indicative of
E. amylovora.
3.1.5.5 Real-time PCR
Based on an evaluation of real-time PCR protocols in the ring tests in 2009 and 2010 (Dreo et al.,
2009; Lopez et al., 2010) the protocol described by Pirc et al. (2009), which targets chromosomal
sequences, was recommended. A duplex real-time PCR based on chromosomal sequences is also
available but has not been ring tested (Lehman et al., 2008).
Real-time PCR according to Pirc et al. (2009)
The following oligonucleotides are used:
Ams116F primer: 5′-TCC CAC ATA CTG TGA ATC ATC CA-3′
Ams189R primer: 5′-GGG TAT TTG CGC TAA TTT TAT TCG-3′
Ams141T probe: FAM-CCA GAA TCT GGC CCG CGT ATA CCG-TAMRA
The reaction is carried out in a final volume of 25 µl. The PCR mixture is composed of: ultrapure
water, 2.5 µl; 2× TaqMan Fast Universal PCR Master Mix (Applied Biosystems1), 12.5 µl; Ams116F
10 pmol/µl, 2.25 µl; Ams189R 10 pmol/µl, 2.25 µl; FAM-labelled Ams141T 10 pmol/µl, 0.5 µl; and
5 µl DNA extract (added to the 20 µl PCR mix). The cycling parameters are: 2 min at 50 ºC; 10 min at
95 ºC; and 40 cycles of 15 s at 95 ºC and 1 min at 60 ºC. The standard mode for temperature ramping
rates on analysers 7900HT and 7900HT Fast (Applied Biosystems1) are: 1.6 ºC/s up and 1.6 ºC/s
down. It is possible to run reactions at slower ramp rates, but with faster ramp rates (up and down at
approximately 3.5 ºC/s) the results were not acceptable. The expected amplicon size is 74 bp.
For analysis of the real-time PCR results, there are usually different options available, automatic or
manual, for setting the signal and noise limits. The instructions for the appropriate software should be
followed. The baseline should be set automatically, and the threshold should be set manually crossing
the exponential phase of the control amplification curves.
The accuracy in the 2010 ring test was 0.80, 0.85 and 0.76 with the DNA extraction method of Llop
et al. (1999), the REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich1) and Taylor et al. (2001),
respectively.
Real-time PCR according to Gottsberger (2010)
The following oligonucleotides that target the E. amylovora chromosome are used:
hpEaF primer: 5′-CCG TGG AGA CCG ATC TTT TA-3′
hpEaR primer: 5′-AAG TTT CTC CGC CCT ACG AT-3′
hpEaP probe: FAM-TCG TCG AAT GCT GCC TCT CT-MGB
The reaction is carried out in a final volume of 20 µl. The PCR mixture is composed of: ultrapure
water, 6 µl; 2× TaqMan Universal PCR Master Mix (Applied Biosystems1), 10 µl; hpEaF 10 pmol/µl,
1 µl; hpEaR 10 pmol/µl, 1 µl; hpEaP 1 pmol/µl, 1 µl; and 1 µl DNA extract (added to the 19 µl PCR
mix). The cycling parameters are: 2 min at 50 °C; 10 min at 95 °C; and 50 cycles of 15 s at 95 °C and
1 min at 60°C. The expected amplicon size is 138 bp.
For analysis of the real-time PCR results, there are usually different options available, automatic or
manual, for setting the signal and noise limits. The instructions for the appropriate software should be
followed. The baseline should be set automatically, and the threshold should be set manually crossing
the exponential phase of the control amplification curves.
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The accuracy of this real-time PCR could not be tested in the 2010 ring test; however, it was tested in
parallel with the real-time PCR of Pirc et al. (2009) by one laboratory and gave the same qualitative
results with the DNA extraction from Llop et al. (1999).
3.1.5.6 Interpretation of results from PCR
Conventional PCR
The pathogen-specific PCR will be considered valid only if:
(1) the positive control produces the correct size amplicon for the bacterium
(2) no amplicons of the correct size for the bacterium are produced in the negative extraction control and the negative amplification control.
If the 16S rDNA internal control primers are also used, the negative (healthy plant tissue) control (if
used), positive control and each of the test samples must produce a 1.6 kilobase (kb) amplicon (16S
rDNA). Note that synthetic or plasmid-positive controls will not produce a 1.6 kb amplicon. Failure of
the samples to amplify with the internal control primers suggests for example that the DNA extraction
has failed, the nucleic acid has not been included in the reaction mixture, compounds inhibitory to
PCR are present in the DNA extract or the DNA has degraded.
The test on a sample will be considered positive if it produces an amplicon of the correct size.
Real-time PCR
The real-time-PCR will be considered valid only if:
(1) the positive control produces an amplification curve with the pathogen-specific primers
(2) no amplification curve is produced (i.e. cycle threshold (Ct) value is 40) in the negative extraction control and the negative amplification control.
If the COX internal control primers are also used, the negative control (if used), positive control and
each of the test samples must produce an amplification curve. Failure of the samples to produce an
amplification curve with the internal control primers suggests for example that the DNA extraction has
failed, the nucleic acid has not been included in the reaction mixture, compounds inhibitory to PCR
are present in the DNA extract or the DNA has degraded.
The test on a sample will be considered positive if it produces a typical amplification curve in an
exponential manner. The Ct value needs to be verified in each laboratory when implementing the test
for the first time.
3.1.5.7 Loop-mediated isothermal amplification
The LAMP protocol was developed and described by Temple et al. (2008) and Temple and Johnson
(2011). It was evaluated in the 2010 ring test because it was considered appropriate for laboratories
not equipped for PCR and it is easy to perform. In the ring test, the LAMP protocol using primers to
detect the chromosomal gene amsL of E. amylovora was found to lack appropriate sensitivity for
analysis of samples with low bacterial populations. Consequently, the LAMP protocol described
below to detect chromosomal amsL is recommended only for the analysis of symptomatic samples
with more than 105–106 c.f.u./ml. The protocol from Temple and Johnson (2011) using primers to
detect pEA29 was not evaluated in the ring test.
The LAMP primers to detect amsL Bare:
ALB Fip: 5′-CTG CCT GAG TAC GCA GCT GAT TGC ACG TTT TAC AGC TCG CT-3′
ALB Bip: 5′-TCG TCG GTA AAG TGA TGG GTG CCC AGC TTA AGG GGC TGA AG-3′
ALB F: 5′-GCC CAC ATT CGA ATT TGA CC-3′
ALB B: 5′-CGG TTA ATC ACC GGT GTC A-3′
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Primers Fip and Bip were used at 2.4 μM and primers F and B at 0.2 μM final concentrations. Melting
temperatures for primers were between 58 and 60 °C. The LAMP reaction mixture is composed of:
10× ThermoPol buffer (New England Biolabs1), 5 µl; dNTPs 10 mM, 5 µl; MgSO4 100 mM, 2 µl;
bovine serum albumin (BSA) 10 mg/ml, 2 µl; ALB Fip 100 μM, 1.2 µl; ALB Bip 100 μM, 1.2 µl;
ALB F 10 μM, 1 µl; ALB B 10 μM, 1 µl; Bst DNA polymerase 8 U/µl, 2 µl; template DNA, 5 µl; and
ultrapure water, 24.6 µl. Note that the Bst DNA polymerase, template DNA and ultrapure water are
not added to the master mix, but are added separately after aliquoting the master mix. Before starting
the LAMP reaction, a water bath or a thermocycler is set at 65 °C. The mix is prepared and 18.4 μl is
pipetted into each individual 0.2 ml PCR tube. The Bst DNA polymerase, template DNA and ultrapure
water are then pipetted separately into each tube with master mix. The tubes are spun down in a plate
spinner (1 000 r.p.m. for 30 s) and are placed in the water bath (65 °C) in a holder so the reaction end
is submerged, or in the thermocycler (65 °C) for 55 min. The tubes are removed and allowed to cool
for 10 s.
The test on a sample is positive if the presence of precipitate as cloudiness in the tube or the presence
of a solid white magnesium pyrophosphate precipitate at the bottom of the tube is observed, as for the
positive control. A clear solution indicates a negative test result, as should be observed for the negative
control.
The accuracy in the 2010 ring test was 0.64, but for samples with 105–106 c.f.u./ml the accuracy was
0.80. For this reason LAMP is recommended only for the analysis of symptomatic samples.
3.2 Detection in asymptomatic plants
The recommended screening tests are indicated in the flow diagram in Figure 2.
3.2.1 Sampling and sample preparation
Asymptomatic samples can be processed individually (preferred) or in groups of up to 100 (EPPO,
2013). Precautions to avoid cross-contamination should be taken when collecting the samples and
during the extraction process. Sampling and sample preparation can be performed following one of the
following protocols:
- Blossoms, shoots, fruitlets or stem segments are collected in sterile bags or containers in summer or early autumn, after favourable conditions for the multiplication of E. amylovora
have occurred and when average temperatures rise above about 15 ºC (van der Zwet and Beer,
1995). Young shoots approximately 20 cm in length, or blossoms when available, are cut from
the suspect plant. If analyses need to be performed in winter, five to ten buds are collected per
plant. In the laboratory, blossoms when available, the peduncle and base of the limb of several
leaves from the base of the shoots, or the stem segments are cut from the selected plants. About
0.1–1.0 g plant material is weighed and macerated in antioxidant buffer following the protocol
described in section 3.1.2.
- A sampling procedure reported but not validated for the analysis of twigs of asymptomatic woody material from nurseries is as follows. A sample comprises 100 twigs, each about 10 cm
in length, from 100 plants. If there are several plant genera in the lot, these should be
represented equally in the sample (with a maximum of three genera per sample). From each
sample 30 twigs are randomly taken and each twig is cut into four pieces (producing 120 stem
pieces). The samples are covered with sterile PBS containing 0.1% Tween 20 in Erlenmeyer
flasks, and the flasks are stirred vigorously on a rotary shaker for 1.5 h at room temperature. The
extract is filtered through filter paper held in a sintered glass filter using a vacuum pump, and
the filtrate is collected. The filtrate is used directly for analysis or centrifuged at 10 000 g for
20 min. The pellet is suspended in 4.5 ml sterile PBS. The detection techniques indicated below
are performed. A similar protocol can be applied for leaves, shoots, flowers and buds.
Depending on the timing of the sampling, the expected recovery of E. amylovora will vary, with
maximum recovery in summer (providing weather conditions are favourable to E. amylovora) and
reduced recovery in winter. Samples should be processed immediately by performing enrichment
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followed by DASI-ELISA, PCR and isolation using the protocols described for each technique for
symptomatic samples in López et al. (2006). Immunofluorescence is optional; if done, it must be done
directly on the extracts, before enrichment.
3.2.2 Screening tests
Direct analysis of asymptomatic samples is normally negative for E. amylovora because of the low
bacterial population. Consequently, when analysing asymptomatic material, it is an absolute
requirement to perform enrichment from samples prepared in the antioxidant buffer (section 3.2.1)
(Gorris et al., 1996) for 72 h at approximately 25 ºC. It is advisable to perform at least two of these
screening tests based on different biological principles:
- Enrichment-isolation. Follow the procedure for symptomatic samples (section 3.1.3.2).
- Enrichment-DASI-ELISA. Follow the procedure for symptomatic samples (section 3.1.4.1).
- Enrichment-PCR or enrichment-real-time PCR. Use 500–1000 µl of the samples enriched in King’s B and/or CCT media for DNA extraction, then follow the procedure for amplification
according to Taylor et al. (2001) or Llop et al. (2000) (section 3.1.5.3) or the real-time PCR
protocols (section 3.1.5.5).
If any of the screening tests are positive but isolation is negative, isolation of the pathogen from the
extract stored at –80 ºC with glycerol or from the enriched samples should be attempted. When three
tests or more are positive and the isolation is negative, it is reasonable to strongly suspect the presence
of E. amylovora in the sample, but identification and confirmation require isolation of the pathogen
from new samples and subsequent identification of the bacterium.
4. Identification
Identification should be based on results obtained from several techniques because other species of
Erwinia such as E. piriflorinigrans (López et al., 2011), E. pyrifoliae (Kim et al., 1999; Rhim et al.,
1999), E. uzenensis (Matsuura et al., 2012) and other Erwinia spp. (Kim et al., 2001a, 2001b; Palacio-
Bielsa et al., 2012) share similar morphological, serological and molecular characteristics to that of E.
amylovora. Differentiation of E. amylovora from these closely related Erwinia species (that can be
found in similarly symptomatic tissues in some hosts) can be achieved with a combination of three
techniques based on different biological principles:
- PCR based on chromosomal DNA (sections 3.1.5.2 and 4.3.1)
- DASI-ELISA using specific monoclonal antibodies as described for detection (section 3.1.4.1, excluding the enrichment step)
- Inoculation into fire blight hosts to fulfil the requirements of Koch's postulates, including re-isolation of the inoculated pathogen (section 4.4).
For identification of colonies, at least two of these three techniques are recommended to be used.
Other tests can also be used depending on the experience of the laboratory; these are described below.
When required, the final confirmation of a culture’s identification should include a pathogenicity test.
The E. amylovora isolates recommended for use as positive controls are NCPPB 683 and CFBP 1430.
The following collections, among others, can provide different E. amylovora reference strains:
National Collection of Plant Pathogenic Bacteria (NCPPB), Fera, York, United Kingdom; Collection
Française de Bactéries Phytopathogènes (CFBP), French National Institute for Agricultural Research
(INRA), Station Phytobactériologie, Angers, France; Belgian Co-ordinated Collection of Micro-
organisms BCCM/LMG Bacteria Collection, Ghent, Belgium; International Collection of
Microorganisms from Plants (ICMP), Manaaki Whenua Landcare Research, Auckland, New Zealand;
and American Type Culture Collection (ATTC), Manassas, VA, United States. The authenticity of the
strains can be guaranteed only if directly obtained from the culture collections.
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4.1 Nutritional and enzymatic identification
Key phenotypic tests are useful and are still used for identification, but it is advised to combine them
with pathogenicity assays and a serological or molecular test. Members of the genus Erwinia are
defined as Gram-negative, facultative anaerobes, motile by peritrichous flagella, rod-shaped, and able
to produce acid from glucose, fructose, galactose and sucrose. Key phenotypic properties (Paulin,
2000) that are common to most strains in E. amylovora, according to the methods of Jones and Geider
(2001), are: oxidase test (–), oxidative/fermentative (O/F) test (+/+), fluorescent pigment in King’s B
medium under UV light (–), levan production (+), nitrate reduction (–), citrate utilization (+), gelatine
liquefaction (+), urease and indol (–) and colony morphology on CCT medium.
The following tests differentiate E. amylovora from E. pyrifoliae and E. piriflorinigrans, although
some physiological and biochemical characteristics may vary for some strains (Table 1).
Table 1. Differences among Erwinia amylovora, Erwinia pyrifoliae and Erwinia piriflorinigrans
Microbiological test Erwinia amylovora Erwinia pyrifoliae Erwinia piriflorinigrans
Gelatin hydrolysis + – –
Inositol† – ND +
Sorbitol† + + –
Aesculin† V – +
Melibiose† – – +
D-Raffinose† – – +
β-Gentiobiose† + – +
Amplification with‡ EP16A/EPI62C CPS1/CPS2C
– + ND
† From Roselló et al. (2006) and López et al. (2011). Oxidation of substrates in API 50 CH strips (bioMérieux)
using the method described by López et al. (2011). More than 90% of strains give the results indicated. ‡ According to Kim et al. (2001b).
ND, not determined; V, variable.
4.1.1 Biochemical characterization
4.1.1.1 Nutritional and enzymatic profiling
Identification of E. amylovora can be obtained biochemically by profiling on the API system 20 E and
50 CH strips (bioMérieux1).
API 20 E1. The manufacturer’s instructions should be followed for preparing the suspension and
inoculating the strip. The strip is incubated at 25–26 ºC. The reading after 48 h for a typical
E. amylovora culture should be as follows: the tests lysine decarboxylase (LDC), ornithine
decarboxylase (ODC), citrate utilization (CIT), H2S production (SH2), urease (URE), tryptophan
deaminase (TDA), indole production (IND) and rhamnose oxidation (RHA) should be negative, while
sucrose oxidation (SAC) should be positive. Other tests may vary by strain, according to Donat et al.
(2007).
API 50 CH1. A suspension of OD 1.0 (at 600 nm wavelength) is prepared in PBS. One millilitre of the
suspension is added to 20 ml Ayers medium (NH4H2PO4, 1 g; KCl, 0.2 g; MgSO4, 0.2 g; bromothymol
blue 0.2%, 75 ml; distilled water, 1 litre; pH 7; sterilized at 120 ºC for 20 min) (Ayers et al., 1919).
The manufacturer’s instructions should be followed for inoculating the strip. The strip is incubated at
25–26 ºC under aerobic conditions. Utilization of the different carbohydrates is observed by the
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development of a yellow colour in the well. The reading after 72 h for a typical E. amylovora culture
should be positive for L-arabinose, ribose, D-glucose, D-fructose, mannitol, sorbitol, N-
acetylglucosamine, sucrose, trehalose and β-gentiobiose. The remaining sugars are not utilized by
E. amylovora in these conditions, but some strains can utilize glycerol and D-fucose, according to
Donat et al. (2007).
4.1.1.2 Automated identification
An automated identification system based on differential results of 94 phenotypic tests in a microtiter
plate and accompanying analysis software are commercially available (OmniLog1, Biolog1). The
manufacturers’ instructions should be followed for presumptive identification of suspected
E. amylovora isolates.
4.1.1.3 Fatty acid profiling
In fatty acid profiling (FAP), levan-positive, non-fluorescent colonies are grown on commercially
available trypticase soy agar at 28 ºC for 48 h (Sasser, 1990). An appropriate fatty acid extraction
procedure is applied and the extract is analysed using the commercially available Sherlock Microbial
Identification System (MIS) (MIDI1) or other appropriate software for presumptive identification of
E. amylovora, according to Wells et al. (1994).
4.2 Serological identification
4.2.1 Agglutination
Suspected E. amylovora colonies can be presumptively identified by slide agglutination. A dense
suspension of cells is mixed with a drop of PBS and a drop of E. amylovora specific antiserum
(undiluted, or at 1:5 to 1:10 dilution only) on a slide. Monoclonal antibodies can be used providing
they agglutinate the reference strains. The specificity of the antibodies must be established in advance.
4.2.2 Immunofluorescence
A suspension of approximately 106 cells/ml is prepared in PBS from levan-positive, non-fluorescent
colonies and the immunofluorescence procedure described in section 3.1.4.3 is followed. The
specificity of the antibodies must be established in advance.
4.2.3 ELISA
Direct tissue print-ELISA (section 3.1.4.2), DASI-ELISA (section 3.1.4.1) and indirect ELISA (see
below) for isolate identification can be performed using specific monoclonal antibodies as described
for detection. A mixture of monoclonal antibodies has been validated in two ring tests for DASI-
ELISA. A suspension of approximately 108 cells/ml is prepared in PBS from suspected colonies. The
DASI-ELISA procedure in section 3.1.4.1 can be used, but without the enrichment step.
Indirect ELISA
Pure cultures of the suspected isolates are treated at 100 ºC for 10 min in a water bath or on a heating
block to reduce non-specific reactions with commercial monoclonal antibodies. Aliquots of 200 µl
culture are mixed with an equal volume of carbonate buffer (Na2CO3, 1.59 g; NaHCO3, 2.93 g;
distilled water, 1 litre; pH 9.6) and this solution is applied to at least two wells of a microtiter plate.
The plate is incubated at 37 ºC for 1 h or at 4 ºC overnight. Extracts are flicked out from the wells and
the plate is washed three times with washing buffer (see the DASI-ELISA protocol). The specific
commercial anti-E. amylovora antibodies from Plant Print Diagnòstics SL1 are prepared at the
recommended dilutions. To each well is added 200 µl of the diluted anti-E. amylovora antibody
solution and the plate is incubated at 37 ºC for 1 h. The antibody solution is flicked out from the wells
and the wells are washed as before. The appropriate dilution of secondary antibody-alkaline
phosphatase conjugate (GAM-AP) is prepared in PBS containing 0.5% BSA. To each well is added
200 µl of the diluted conjugate antibody and the plate is incubated at 37 ºC for 1 h. The conjugated
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antibody is flicked out from the wells and the wells are washed as before. A 1 mg/ml alkaline
phosphatase substrate (p-nitrophenylphosphate) is prepared in substrate buffer (diethanol amine,
97 ml; 800 ml distilled water; adjusted to pH 9.8 with concentrated HCl; then the volume is adjusted
to 1 000 ml with distilled water). To each well is added 200 µl alkaline phosphatase substrate solution.
The plate is incubated in the dark at room temperature and read at 405 nm at regular intervals within
90 min. A positive test is indicated by substrate conversion to a yellow colour.
4.2.4 Lateral flow immunoassay
A suspension of 107 c.f.u./ml of the pure culture is prepared for presumptive identification. Buffers
and procedures provided by the manufacturers of the kits are used, as described in section 3.1.4.4.
4.3 Molecular identification
4.3.1 PCR
A suspension of approximately 106 cells/ml is prepared in molecular grade sterile water from purified
levan-positive, non-fluorescent colonies and is treated at 100 ºC for 10 min. The appropriate PCR
procedures or the LAMP protocol are applied as described in sections 3.1.5.2 to 3.1.5.4 (directly,
without DNA extraction). When using PCR to identify isolated colonies, 1 U of Taq DNA polymerase
should be used (instead of 2 U as for plant material).
4.3.2 Macro-restriction and pulsed field gel electrophoresis
Pulsed field gel electrophoresis (PFGE) analysis of genomic DNA after XbaI digestion according to
Jock et al. (2002) shows six patterns for E. amylovora European strains. The method can provide
useful information for strain differentiation and has been applied to understanding the spread of fire
blight in Europe (Jock et al., 2002; Donat et al., 2007).
4.4 Pathogenicity techniques
Suspected E. amylovora colonies should be inoculated back into host plants to fulfil Koch’s postulates
and verify their pathogenicity. For plant inoculation, susceptible cultivars of pear (e.g. Conference,
Doyenne du Comice, Williams, Passa Crassane), apple (e.g. Fuji, Gala, Idared, Jonathan), loquat (e.g.
Algerie, Tanaka), Crataegus spp., Cotoneaster spp. or Pyracantha spp. are used. Young shoots are
inoculated by cutting across a young leaf through the central vein with scissors dipped in a
109 c.f.u./ml suspension of each isolate prepared in PBS. The plants are maintained at 20–25 ºC at
approximately 80% relative humidity for one to two weeks. Detached young shoots that have been
surface-sterilized (treated with 70% ethanol for 30 s then washed three times with sterile distilled
water) from greenhouse-grown plants can also be inoculated in the same way and kept in tubes with
sterile 1% agar. The tubes should be kept at 20–25 ºC with 16 h light per day.
Inoculation can also be performed on detached immature fruits of susceptible cultivars of pear, apple
and loquat by placing 10 μl of 109 c.f.u./ml suspensions of the isolates in PBS into a fresh wound on
the surface of disinfected fruits (treated with 70% commercial chlorine for 30 min then washed three
times with sterile distilled water). The fruits should be incubated in a humid chamber at 25 ºC for three
to five days.
E. amylovora-like colonies are re-isolated and characterized from inoculated organs showing typical
fire blight symptoms. A positive test is evident by the oozing of bacteria and browning around the
inoculation site after two to seven days, as seen in the positive E. amylovora control, providing no
lesions are or only a small necrotic lesion is observed at the wound site in the negative control.
Other inoculation techniques are possible. Hypersensitive reactions in tobacco leaves may indicate
expression of the hrp genes of E. amylovora, but this test may be positive for many other plant
pathogenic bacteria. Tobacco plants of cultivars Xanthi or Samsun with more than five to six leaves
should be used. Bacterial suspensions of 109 c.f.u./ml (OD at 600 nm, 1.0) are prepared and a needle
and syringe used to inject the suspensions into the intracellular space of mature leaves. Complete
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collapse of the infiltrated tissue after 24–48 h at room temperature is recorded as positive, as observed
in the positive E. amylovora control.
5. Records
Records and evidence should be retained as described in section 2.5 of ISPM 27 (Diagnostic protocols
for regulated pests).
In cases where other contracting parties may be affected by the results of the diagnosis, in particular in
cases of non-compliance (ISPM 13 (Guidelines for the notification of non-compliance and emergency
action) and where the pest is found in an area for the first time, the following records and evidence and
additional material should be kept for at least one year in a manner that ensures traceability: the
original sample, culture(s) of the pest, preserved or slide-mounted specimens or test materials (e.g.
photographs of gels, ELISA plate results printouts and PCR amplicons).
6. Contact Points for Further Information
Further information on this protocol can be obtained from:
Centro de Protección Vegetal, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera
Moncada-Náquera km 4.5, 46113 Moncada (Valencia), Spain (María M. López; e-mail:
[email protected]; tel.: +34 963424000; fax +34 963424001).
Plant Health and Environment Laboratory, Investigation and Diagnostic Centres, Ministry for Primary
Industries, 231 Morrin Road, St Johns, Auckland 1140, New Zealand (Robert Taylor; e-mail:
[email protected]; tel.: +64 99093548; fax: +64 99095739).
A request for a revision to a diagnostic protocol may be submitted by NPPOs, regional plant
protection organizations (RPPOs) or Commission on Phytosanitary Measures (CPM) subsidiary bodies
through the IPPC Secretariat ([email protected]), which will in turn forward it to the Technical Panel on
Diagnostic Protocols (TPDP).
7. Acknowledgements
The first draft of this protocol was written by M.M. López (Centro de Protección Vegetal, IVIA, Spain
(see preceding section)) and revised by R. Taylor (Plant Health and Environment Laboratory,
Investigation and Diagnostic Centres, Ministry for Primary Industries, New Zealand (see preceding
section)) and R. Roberts (Tree Fruit Research Laboratory, USDA-ARS, United States).
Most techniques described were ring tested in a DIAGPRO project financed by the European Union in
2003, in an EUPHRESCO project in 2009 and in a Spanish project in 2010.
8. References
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